Surface Invader

A large part of the human genome is conserved between individuals meaning that our individualities occur from a few, respectively speaking, varying sequences known as polymorphisms. While there are several types of conserved polymorphisms found within an individual’s genetic makeup 90% of them are single nucleotide polymorpshisms (SNPs). A SNP is a conserved sequence containing single base pair alternatives, occurring every 500 ?1000 base pairs, leading to differences in disease predisposition as well as normal biological makeup. The ability to effectively detect and score these polymorphisms has offered fresh insights into human pathophysiology; leading to new applications in areas such as population genetics, drug development, forensics, as well as the study of cancer and genetic-related diseases. Thus an accurate and high-throughput means of SNP scoring has become increasingly important.

Traditionally genetic studies relied on the mapping of short tandem repeat markers (STRMs) because of their high degree of heterozygosity and the amount of information per marker. A drawback to this method is that electrophoretic separations are required to separate the large number of alleles. SNP analysis has proven more amenable for automated, multiplex analysis, with results that are more accurate and reliable than its STRM counterpart. Whole genome amplification methods have been successfully used to genotype more than 100,000 SNPs on a pair of arrays. The use of molecular inversion “padlock?probes has also proven an efficient method of scoring more 1000 SNPs in a single assay.

The invader assay, a focus of our group, is a method of SNP scoring that eliminates the need for amplification of sample DNA and has been shown to effectively work in multiplexed assay formats. In the invader reaction, three single-stranded DNA chains form a ternary complex, the invasive structure, having a one base-pair overlap. This complex is composed of a DNA target molecule, which contains the SNP sequence of interest, and two other oligonucleotide sequences: an upstream invader and downstream probe. These three oligonucleotide strands hybridize to one another, forming a one base-pair junction causing the 5?end of the probe oligonucleotide to form a “flap.?This flap is then cleaved by a flap endonuclease (FEN) enzyme. In the analytical assay, enzymatic cleavage separates a FRET pair allowing donor fluorescence to be detected as well as monitored in real-time. It has been shown that the cleavage rates are 300 times higher when the probe sequence is complementary at the polymorphic base than when it is not. A major advantage of the invader assay is its sensitivity, demonstrated by the ability to score SNPs on non-amplified genomic DNA. This precludes the possibility of false readings and confusion from contamination, which can occur during PCR. In comparison to PCR-RFLP, the invader method cuts the total time of sample preparation and analysis by over 20 hours, reduces the number of hands on steps from 12 to 4, and excludes the need for thermal cycling saving both time and money.  

Our group has adapted the invader reaction to a surface-based platform to meet the goal of a high-throughput system capable of scoring many of SNPs simultaneously. To achieve this, the upstream invader and downstream probe oligonucleotides are attached to the surface on which the invader reaction occurs. Parallelization of the surface invader assay has no perceptible effect on the overall sensitivity and thus the assay is capable of detecting multiple polymorphisms directly from non-amplified genomic DNA. A universal mode of detection was developed to facilitate parallelization of the invader assay in the array format. This detection involves ligation of a primer for rolling circle amplification (RCA) directly to the cleaved probe. One primer can be ligated to all possible probe sequences because of the use of a degenerate template DNA. Once the primer is ligated to the probe, it is extended by RCA and labeled with a double-strand specific fluorescent dye.  

We have shown that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of SNPs directly from unamplified genomic DNA target. In addition, we created a “universal?mode of detection that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high density arrays able to analyze hundreds of thousands or even millions of SNPs.  

There are many new possibilities for further development of the invader assay. Currently we are focusing on the areas that we feel are the most important for the creation of a truly effective SNP detection method:
- Development of new substrates: Silicon, diamond and glassy carbon surfaces have advantages over glass for the construction of biomolecule arrays. The primary advantage is the nature of the chemical bond between the substrate and the linker. The instability of biomolecule arrays constructed on glass is well known. This stems from the relatively weak Si-O chemical linkage on the surface. In contrast, linkage on silicon is formed by a strong Si-C bond and linkage on diamond and glassy carbon if formed by an even stronger C-C bond. Surface stability is of utmost importance in the invader reaction where high temperatures (60C) and long reactions times (3h) are common.
- New Compounds for Gold Self-Assembled Monolayers: Self-assembled monolayers on gold are arguably the second most common platform for construction of biomolecule arrays. These SAMs are reasonably stable to the conditions of the invader reaction. For some time, we have used an oligonucleotide attachment chemistry that requires the DNA to have a reactive sulfhydryl. However, such DNAs are inconvenient to use because significant preparation is required. To combat this problem, we successfully designed, synthesized and tested two new compounds that are reactive to primary amine containing DNA. These new compounds form excellent self-assembled monolayers on gold. And, amine containing DNA are much simpler to prepare.
- Photolithographically Prepared Arrays: Currently oligonucleotide attachment methods involve the spotting of oligonucleotide solutions onto the surface of interest. This mode of attachment is impractical for fabrication of complex high-density arrays because the synthesis, purification and handling of so many different oligos simply isn’t feasible. Our solution to this problem is to generate arrays for the surface invader reaction using photolithography. We are working in close collaboration with Franco Cerrina at the University of Wisconsin Center for Nanotechnology to develop a maskless array synthesizer (MAS) instrument that is capable of growing two different oligos of opposite polarity on a single array element. This advance holds the key for large scale implementation of the surface invader assay.

  


Here are some helpful references that give a better background:
(1) Lu, M., et al. J. Am. Chem. Soc. 2001, 124, 7924 ?7931.
(2) Lyamichev, V., et al. Nature 1999, 17, 292 ?296.
(3) Landegren, U., Nilson, M., Kwok, P.Y. Genome Research 1998, 8, 769 ?776.
(4) Brookes, A.J. Gene 1999, 234, 177 ?186.
(5) Chen, Y.; Shortreed, M. R.; Peelen, D.; Lu, M.; Smith, L. M. J. Am. Chem. Soc. 2004, 126, 3016.
(6) Wang, D.G., et al. Science 1998, 280, 1077 ?1082.